Research Report

Research Progress of Woody Plant Regeneration System  

WonJin Han 1,2 , Shenkui Liu3 , yuanyuan Bu1,2
1 Key Laboratory of Saline-Alkali Vegetation Ecology Restoration (Northeast Forestry University), Ministry of Education, Harbin 150040, China
2 College of Life Science, Northeast Forestry University, Harbin 150040, China
3 The State Key Laboratory of Subtropical Silviculture, Zhejiang Agriculture and Forestry University, Lin’An, Zhejiang 311300, China
Author    Correspondence author
Molecular Soil Biology, 2020, Vol. 11, No. 1   doi: 10.5376/msb.2020.11.0001
Received: 20 Jan., 2020    Accepted: 10 Feb., 2020    Published: 28 Feb., 2020
© 2020 BioPublisher Publishing Platform
This is an open access article published under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Preferred citation for this article:

Han W.J., Liu A.I. and Bu Y.Y., 2019, Research progress of woody plant regeneration system, Molecular Soil Biology, 11(1): 1-7 (doi: 10.5376/msb.2020.11.0001)


The woody plant regeneration is very beneficial not only for the rapid propagation or preservation of valuable woody plants, but also for the establishment of genetic transformation system. In general, the regeneration is occurred either directly from explants or indirectly through the callus. In both cases, there exists a lot of factors and conditions which influence significantly on the regeneration process. Among them, different genotypes, types of explants and exogenous plant hormones are especially important. In addition, the effects of the cultural temperature, medium compositions and other factors can not be ignored. In this review, the research advances and several influencing factors in the field of woody plant regeneration are summarized in detail.

Plant regeneration; Genetic transformation system; Callus; Plant hormones


If some part of a plant is injured, plant has a high capacity to regenerate new tissues and organs to reduce the damage as much as possible (Pulianmackal et al., 2014). The tissue culture in which the whole plants can be developed from small tissues or cells in vitro has been developed from the early 20th century. The common in vitro plant regeneration, that is, the ectopic meristems are induced first from explants and they develop into shoots and roots forming the whole organisms, has been widely used in rapid propagation and genetic transformation of plants in recent years (Momoko et al., 2016).


In the past decades, the regeneration system has been established in a lot of woody plant species for rapid propagation, preservation of valuable species and genetic transformation. For example, in several Salix species, which were well-known with salt, drought resistant woody plants, the genetic transformation systems were also established based on in vitro plant regeneration, providing a broad way for exploring important stress resistant genes (Qing et al., 2017). In 2005, the medical woody plant Phellodendron amurense was also in vitro regenerated for its medical value (Azad et al., 2005).


There are a lot of factors that have considerable effects on the regeneration process.


They have combinational effects each other in order to cause the dedifferentiation of somatic cells. In this review, based on the research achievements in the field of woody plant regeneration, several important influencing factors on the regeneration process were discussed.


1 Effect of genotype

The induction of embryo and shoot organogenesis in vitro plant regeneration is largely influenced by genotype.  The different culture conditions in different species are required because of quantitative or qualitative genetic varieties. Therefore, it is very important to select appropriate plant species which are competent for in vitro cultures (Henry et al., 1994). Nermin et al. (1998) showed that the levels of adventitious shoot regeneration in nine Populus tremula genotypes were mainly due to the genetic differences.  In five willow clones, the shoot multiplication, elongation and rooting ability was also related to genotype (Lotta et al., 1985). In additions, the regeneration rates in five Populus genotypes were 97, 94.7, 72.3, below 50% respectively in the same cultural condition (Yao et al., 2007). This kind of trend can also be seen in Populus deltoides and Populus tremuloides (Ahuja et al., 1983; Gary et al., 1989). In the somatic embryogenesis of five Maritime Pine genotypes, the percentage of induction was also strongly influenced by genetic differences (Isabel et al., 2019). It has been reported that the genes related to phytohormone signals and several protein kinases are involved in regeneration process (Henry et al., 1994).  It has also been shown that there exist some specific genes taking part in every steps of shoot organogenesis including dedifferentiation, acquisition of competence, induction of ectopic apical meristem. Depending on different genotypes, genes related with plant regeneration could not be expressed in different unsuitable media (Xiu et al., 2008). Depending on where explants come from, or the genotype of original plant, the ability of regeneration varies significantly. In the same cultural condition, among 16 different genotypes of populus deltoides, for example, 6 types were difficult to induce, another four kinds were over 50%, the rest was from 3-23%, indicating the considerable differences depending on genotypes (Coleman et al., 1989).  


2 Choice of Explant

Theoretically, all kinds of plant cells have totipotency.  However, research showed that , even in one plant, different parts or different tissues had considerable differences in the capacity to regenerate. Therefore, it is necessary to select appropriate explants for experiment (Zhang et al., 2004). In Luis et al. (1999) experiment, hypocotyls, cotyledons, cotyledonary nodes and primary leaves were used as explants in Eucalyptus grandis x E.urophylla, and the percentage of shoot induction was highest in cotyledonary nodes-derived calluses, and this was mainly because of meristem existence in the original explant inducing calli with higher regeneration capacity  (Luis et al., 1999). In the same concentrations of plant hormones, the abilities of callus and shoot induction in Populus Euramericana were low in leaves, comparing with petioles and stem segments, indicating that different parts of plant have different regeneration abilities (Sun et al., 2004). Even, in the same organ of a plant, different parts are not all same in the regeneration capacity. The differentiation study of Populus davidiana X P. bolleana Loucne showed that shoot induction rates of lower sections of leaves were higher than middle and upper parts (Chen et al., 1980). In the regeneration of Caesalpinia bonduc, callus induction and number of shoots were better when pulvinus was used as explant, comparing with internode and leaf. The browning of the medium, caused by oxidation of polyphenolic oxidases and peroxidases which lead to cell death, was also less in pulvinus than other explants (Meena et al., 2012). The regenerative abilities of explants are significantly different depending on the ages, generally reducing as plants get older. In the Castanea sativa and Quercus sp. species, although the addition of auxin reactivated cell proliferation, new root meristems did not occur in mature explants (Ballester et al., 1999; Vidal et al., 2003). Other researches also showed that the shoot organogenesis occurred more efficiently in the explants of young plants than mature plants (Dong et al., 1991; Baker and Bhatia, 1993; Becerra et al., 2004; Zhang et al., 2015). This may be because of the lower responsiveness in older plants to plant hormones (Momoko et al., 2016).


3 Plant Hormones

The tissue culture or plant regeneration have been developed rapidly after the discovery of two kinds of plant hormones, called auxin and cytokinin respectively. It is generally known that root is generated in case of high ratios of auxin to cytokinin, while high ratios of cytokinin to auxin cause the shoot induction (Skoog and Miller, 1957). In the regeneration process, how to select the different kinds and concentrations of plant hormones are very important. In general, auxin causes cell proliferative proliferation, while cytokinin makes cells differentiate. GA3 inhibits the occurrence of embryoid bodies (Taras et al., 2019). Plant hormones influencing regeneration process can be divided into endogenous and exogenous hormones, and the kinds and concentrations of endogenous hormones existed in explants vary depending on the type of explants (George et al., 2008). Then, how do auxin and cytokinin added into medium works? Plant growth hormones added in medium cause the expression of some genes (transcription factors), which consequently regulate the further stages of plant regeneration such as shoot initiation, callus formation and so on. For example, cytokinin induces multicomponent phosphorelay signaling system, and the sensory His kinase serves as a cytokinin receptor here. A group of genes participating in this process has not been identified in detail (Saurabh et al., 2015). Many reprogramming regulators(genes) are not expressed in normal cases, but they are activated by wound stress or plant hormones (Momoko et al., 2019).  It has also been reported that auxin leads to activation of cyclin-dependent kinase complexes. Cell reprogramming process in which nuclei size is increased and stabilized for reactivating division also requires auxin in the culture medium. It has also been proved that media with hormones can causes the expression of cell-reprogramming- related hormones, such as WUS, LEC 1,2, BBM so on (Taras et al., 2019). Some experiments also showed that auxin activated specific genes promoting embryonic development, or suppressed these gene activating different kinds of genes instead to inhibit development depending on time and space specificity (Cui et al., 2000).


2,4D is one of the most important hormones for callus induction. Dedifferentiation process in a lot of plant species requires 2,4D. Especially, monocotyledon plants require high concentrations of 2,4D, while in double cotyledon plants, 2,4D requirement is only monocotyledon’s 1/10-1/20. However, after dedifferentiation happens, it must reduce or remove 2,4D in time. It was proved that if 2,4 was not removed in time after embryonic callus formation, it showed that the normal development of embryonic cells did not happen properly. It was also shown that monocotyledon plant not only needed high concentrations of 2,4 D, but also required the combination of 2,4 D and KT (Cui et al., 2000). How does it works?  LEC1 gene, a kind of embryogenic gene, was expressed seven folds in high 2,4D concentration than lower one. Furthermore, more relaxed chromatin was observed in the medium with high concentration of 2,4D (Taras et al., 2019).  In Daucus carota L. cell culture, some kinds of polypeptide and proteins were specifically expressed after 2,4 addition, while they were disappeared after removal of 2,4D (Cui et al., 2000). Experiments also showed that the concentrations of 2,4D had a relationship with DNA methylation.  In Daucus carota L suspension culture, in high 2,4D concentration, the degree of methylation increased, gene expression was also reduced. However, in case of removal of 2,4, the DNA methylation was also not enough, and reactivated the expression of genes again (Cui et al., 2000).


TDZ is also an important man-made most active cytokinin-like material for woody plant regeneration. It has been proved that TDZ leads to the callus formation in several species, and the cell proliferation by TDZ is higher than other growth regulators (Murthy et al, 1998). It has been reported that the range of TDZ used in woody plant regeneration is from 1 to 20uM. Higher than 1uM, it activates the callus formation, adventitious shoots, and somatic embryos and 1-50uM TDZ can activate black wulnut callus production. Prior culturing with TDZ in general has no or little effect on consequent rooting of microshoots.  Cotyledons from mature and immature seeds or leaf tissues are commonly used as explants with TDZ cultivation in several woody plants (Murthy et al. 1998). Meyer et al. (1988) showed that buds induced by TDZ did not elongate and develop into whole plant. It is recommended that TDZ concentration should be low as much as possible, and the explant must be placed on TDZ-adding medium for the shortest time depending on the plant species (Meyer et al., 1988). For example, it was reported that the exposure time of explant to TDZ was maximum 8 weeks (Lu, 1993). 


In regeneration process, the types and concentrations of auxin and cytokinin , and how can combine them are totally determined based on explants.  The effect of different kinds of plant hormones on the callus formation of Salix viminalis L was observed. Regard of this experiment, the most effective auxin was NAA, and then 2,4D, IBA, IAA respectively, while the concentration range was from 0.1-0.5mg/l. In case of cytokinin, 6BA and TDZ was relatively efficient, the range was from 0.5-1mg/l. It was proved that if 1.0mg/l 6BA combined with appropriate concentrations of auxin, especially NAA, the callus induction rate increased significantly (Zhang et al., 2007). This trendy is of course different in other plant species. Azad et al. (2005) showed that NAA was better than IBA in shoot differentiation of Phellodendron amurense Rupr, and the high concentrations of cytokinin and auxin inhibited the formation of shoot. On the other hand, TDZ was more effective than BAP in callus induction. In this study, the most efficient combination of cytokinin and auxin was 1.5uM BAP and 1.0uM NAA (Azad et al., 2005). In five different salix.Spp genotypes, on the other hand, at the same concentration of 6BA(10-6 M), the axillary shoots rate were 4.3,1.3,1.5,1.6,2.0 respectively (Lotta et al., 1985). Cytokinin is essential for DNA synthesis and cell division, but depending on plant species, effective kinds and concentrations are also different. For instance, in Salix tetrasperma Roxb, the most suitable condition of shoot induction was 6BA, 5.0uM. However, more than 6 weeks exposure to 6BA led to hyperhydricity. On the other hand, it was shown that Kn was inferior than BA, which has been also reported in many woody plants (Ahmad et al., 2008; Anis et al., 2010). The efficient effect of 6BA on multiple shoot bud differentiation has also been shown in many other species in different types of explants (Anis et al., 2009; Husain and Anis, 2009; Siddique and Anis, 2009). Multiplication and proliferation after shoot induction was efficient in low BA concentration. This might be because of the residual cytokinin existed in the shoots in the multiplication step. For example, in salix nigra culture, induced shoot buds moved to the 1/10th concentration of PGRs of callus-induced medium for the continuous growth (Lyyra et al., 2006), indicating that lower concentration of 6BA was beneficial for shoot further growth and proliferation. In populus, in the same way, if the concentration of auxin did not reduce in the transition stage from callus to shoot, explants lost their differentiation ability (Zhang, 2003).


The root in general can be induced by adding auxin alone, especially IBA is best. In the root elongation different from root induction, auxin has an inhibitory effect. Therefore, in the woody plant rooting, root induction is generally carried out in auxin-containing medium at first, and then move to the medium without any growth regulator (Lyyra et al., 2006). Azad et al. (1999) showed that among NAA, IBA, IAA, IBA was best in rooting while IAA was worst. These results were same in other medicinal plants, such as Adhatoda vasica and Holo-stemma ada-kodien (Martin, 2002).


4 Other factors

4.1 Medium effects

Cultural medium is also one of the important influencing factors. Zhang et al. (2007) showed that among MS, B5, WPM, improved SH medium, MS medium was best in willow callus induction (92%), while B5 was worst (16%). In Salix exigua regeneration through callus, on the other hand, among WPM, MS, DCR medium, WPM was best in callus induction, while shoot production was best from the calli induced on MS medium. The differences in callus induction according to media were also reported in other woody plants (McCown and Sellmer, 1987), and this might be because of differences in the organic ion concentrations of media.  It was generally known that low salt concentration was preferable for culture in several tree species (Michael et al., 1989).


4.2 Light effect

In the callus induction of salix, in case of no light, the percentage of callus induction was low, and the size was also small. Moreover, color was not green.  Under the light condition, callus color changed into green again (Zhang et al, 2007).  In the apple regeneration using leaves, on the contrary, dark condition promoted regeneration, while light inhibited the regeneration process. In general, red light inhibits adventitious buds formation, while far-red light promotes the formation of adventitious buds. Because red light emitted by incandescent lamp is more than far red light, the induction of adventitious buds might be inhibited under the direct culture of lamp (Shi et al., 2004). It is known that at least two photoreceptors take part in the light response of shoot regeneration, one is blue/UV light receptor which prevent the shoot regeneration, the other is far-red light receptor which defend the explants from the initial light inhibition (Shi et al., 2004).


4.3 Some additional components used in plant regeneration

Some additional components, such as antioxidants, are also used for promoting the regeneration process. For example, in woody plant tissue culture, antioxidant, such as PVP, was also used for reducing oxidation, thus preventing browning in culture (Gupta et al., 1980; Tyagi et al., 1981; Zhong et al., 1995). In the pollen embryogenesis in Datura innoxia, under the treatment of PVP, number of embryos induced increased considerably, and the result was most obvious at 0.5% level, that is, 6 times than control. PVP is known to enhance the enzyme stability by removing phenolics (Loomis and Battaile, 1966). Phenolic compound accumulated in culture is known to inhibit the in vitro growth and morphogenesis (Fridborgcrat et al., 1978). In banana tissue culture, the addition of ascorbic acid to medium prevented the exudation of phenols (Strosse et al., 2004), while shoot multiplication recorded best results in Lawsonia inermis if supplemented with ascorbic acid. Similarly, addition of cysteine to the growth media reduced blackening of explant in banana tissue culture (Strosse et al., 2004). Activated charcoal added in medium also prevented phenolics leaching which prevent the regeneration of Celastrus paniculatus and C. orchioides (Sharada et al., 2003; Prajapati et al., 2003). In Eucalyptus regeneration, the activated charcoal also promoted shoot elongation and made leaves dark green and vigorous (Dibax et al., 2005). If antioxidants, such as glutathione 15-25mg/l, L-cystein 15mg/l PVP 50mg/l, or aminooxyacetic acid 10mg/l were added, the multiple shoot formation was also enhanced in Holostemma ada-kodien Schult (Azad et al., 2005).



This work was supported by the State Key Laboratory of Subtropical Silviculture (KF201707) awarded to Yuanyuan Bu, and further supported by the Program for Changjiang Scholars and Innovative Research Team in University (No. IRT17R99) awarded to Shenkui Liu. The funders had no role in study design.



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